Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E 2 (PGE 2 ). However, little is known about the molecular organization of the TRPV1 phosphorylationcomplexandspecificallyaboutscaffoldingproteinsthatpositiontheproteinkinaseA(PKA)holoenzymeproximaltoTRPV1foreffective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in ϳ80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-( p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE 2 decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE 2 effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (⌬36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in ⌬36 mice. The PGE 2 /PKA signaling defect in DRG neurons from ⌬36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE 2 -induced thermal hyperalgesia was significantly diminished in ⌬36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE 2 /PKA signaling pathway.

SUMMARY Regulation of neuronal excitability and cardiac excitation-contraction coupling requires proper localization of L-type Ca2+ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming CaV1.2 subunit, to foster its surface expression. Disruption of α-actinin function by dominant negative or shRNA constructs reduces CaV1.2 surface localization in HEK293 and neuronal cultures, and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca2+ influx through L-type channels but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca2+-binding deficient calmodulin mutant does not affect basal CaV1.2 surface expression, but inhibits its internalization upon Ca2+ influx. We conclude that α-actinin stabilizes CaV1.2 at the plasma membrane, and that its displacement by Ca2+-calmodulin induces Ca2+-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca2+ influx.

A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.Ligand-gated ion channels (LGICs) 1 mediate the fast inhibitory and excitatory responses of neuronal and muscle cells to neurotransmitters. A universal feature of the type of "Cys-loop" class of LGIC is a common topology of four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconservated cysteinebridge motive (1). In vertebrates this "Cys-bridge" family of phylogenetically related genes codes for cation channels activated by acetylcholine and serotonin or for anion channels activated by GABA and glycine (1). In addition, glutamateand serotonin-gated anion channel genes are known in invertebrates (2, 3). Recently, genes for histamine-gated chloride channels and GABA-gated cation channels were identified in invertebrates (4 -7). The molecular basis of further channel types like acetylcholine-gated chloride channels in invertebrates is, however, still unknown (8). Information from the Drosophila melanogaster genome sequencing project allows identifying all members of the superfamily of ligand-gated ion channels occurring in this species by bioinformatic analysis of new homologous genes. The summarized data obtained from several published bioinformatic analyses (5,6,9,10) show that the group of ligand-gated "chloride" channels consists of 12 genes that are coding for GABA, histamine, and glutamate receptors or new, homologous ion channel types. Four members of this group cannot be directly assigned to the GABA, glutamate, or histamine branches and thus code for putative new types of ligand-gated chloride channels with yet unknown function. In a systematic expression approach of these predicted novel types of ion channels in Xenopus oocytes, it was found that none of the typical neurotransmitters activated these novel types of channels (6). Therefore, we extended the molecular biological analysis of the mRNA and found that the gene CG6112 encodes fo...

The relative contribution of ␣4␤2, ␣7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the ␣7 nAChR agonist. ABBF bound to ␣7 nAChR in rat brain membranes (K i ϭ 62 nM) and to recombinant human 5-hydroxytryptamine (5-HT) 3 receptors (K i ϭ 60 nM). ABBF was a potent agonist at the recombinant rat and human ␣7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT 3 receptor and ␣3␤4, ␣4␤2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3-1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the ␣7 nAChR antagonist methyllycaconitine at 10 g, indicating that it is mediated by ␣7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3-1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3-30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the ␣7 nAChR and that selective ␣7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that ␣7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.Nicotine enhances cognitive functions, such as attention, learning, consolidation, and retention, in both animals and humans, through activation of brain nicotinic acetylcholine receptors (nAChRs) (Levin et al., 1999(Levin et al., , 2006. These ligandgated ion channels are homopentamers formed by five identical subunits (␣7 nAChR) or heteropentamers of multiple ␣ and ␤ subunits. Various isoforms of these subunits have been identified (␣2-␣10; ␤2-␤4; for reviews, see Paterson and Nordberg, 2000;Gotti et al., 2006). The most common nAChRs found in the brain are the ␣7 subtype with a low affinity for nicotine and the ␣4␤2 subtype with a high affinity for nicotine. Evidence from neuroanatomical, electrophysiological, and behavioral studies support a role for both of these receptor subtypes in processes of learning and memory.

BackgroundPhysiologically-based pharmacokinetic (PBPK) modeling has received growing interest as a useful tool for the assessment of drug pharmacokinetics by continuous knowledge integration.ObjectiveThe objective of this study was to build a ciprofloxacin PBPK model for intravenous and oral dosing based on a comprehensive literature review, and evaluate the predictive performance towards pediatric and geriatric patients.MethodsThe aim of this report was to establish confidence in simulations of the ciprofloxacin PBPK model along the development process to facilitate reliable predictions outside of the tested adult age range towards the extremes of ages. Therefore, mean data of 69 published clinical trials were identified and integrated into the model building, simulation and verification process. The predictive performance on both ends of the age scale was assessed using individual data of 258 subjects observed in own clinical trials.ResultsCiprofloxacin model verification demonstrated no concentration-related bias and accurate simulations for the adult age range, with only 4.8% of the mean observed data points for intravenous administration and 12.1% for oral administration being outside the simulated twofold range. Predictions towards the extremes of ages for the area under the plasma concentration–time curve (AUC) and the maximum plasma concentration (Cmax) over the entire span of life revealed a reliable estimation, with only two pediatric AUC observations outside the 90% prediction interval.ConclusionOverall, this ciprofloxacin PBPK modeling approach demonstrated the predictive power of a thoroughly informed middle-out approach towards age groups of interest to potentially support the decision-making process.Electronic supplementary materialThe online version of this article (10.1007/s40262-018-0661-6) contains supplementary material, which is available to authorized users.